Synaptogenesis between neurons is a key mechanism underlying the generation, maintenance, and plasticity of neuronal circuits. The molecular events that govern the development of synapses between neurons are poorly understood. In C. elegans, neurons form stereotypical synapses with each other, providing an ideal model system to study this question. We are studying the synapses formed between the HSNs and VC4,5 neurons that regulate egg-laying behavior. Presynaptic vesicles in the HSN motor neurons were labeled with SNB-1-YFP (Nonet, 1999). Postsynaptic sites in VC4,5 neurons were labeled with LIN-10-RFP (Rongo et al. 1998). Using these markers, two clusters of synapses can typically be visualized in the vulva region of adult animals. We used these markers to examine the formation of synapses during development. SNB-1-YFP and LIN-10-RFP are expressed by the L2 larval stage, but the HSN to VC synapses are not mature until the adult. The clustering of presynaptic vesicles in HSN is first apparent in early L4 larva. When the vesicles first cluster, postsynaptic LIN-10 in the VC4,5 neurons has a homogenous staining pattern. Later in the L4 stage, LIN-10 forms puncta along the VC processes, which resolve to locations next to presynaptic vesicles in early adult. These results suggest that presynaptic specializations precede postsynaptic specializations, at least those marked by LIN-10. We then asked what determines the location of the synapses. We found that the vulva may induce synaptogenesis between these neurons. In
dig-1 animals whose vulvae are anteriorly displaced, the synapses between HSN and VC4,5 were anteriorly displaced accordingly. In
unc-40 and
unc-6 mutants, HSN axons do not contact the vulva because of axon guidence defects. HSN presynaptic vesicles showed abnormal clustering in these mutants. Vulvaless animals resulting from ablation of vulval precursor cells also showed abnormal HSN presynaptic clustering. These results suggest that the developing vulva induces synapse formation between HSN and VC4,5, consistent with its known role in inducing HSN and VC branching. Presynaptic vesicle clustering and synaptic transmission are not essential for the assembly of the postsynaptic apparatus. In
unc-104 mutants, presynaptic vesicles are trapped in the HSN cell body, but postsynaptic clustering of LIN-10 was normal. In
unc-13 mutants, synaptic transmission is blocked, but both LIN-10 localization in VC4,5 and SNB-1 localization in HSN was normal. We are using these markers to identify and characterize mutants with defects in synapse formation, and have isolated a mutant that shows ectopic HSN synapses in the vulval area. Reference: Nonet, M.L. (1999). Visualization of synaptic specializations in live C. elegans with synaptic vesicle protein-GFP fusions. J. Neurosci.Methods 89, 33-40 Rongo C, Whitfield CW, Rodal A, Kim SK, Kaplan JM. (1998) LIN-10 is a shared component of the polarized protein localization pathways in neurons and epithelia. Cell. 94:751-9.